a) Field of the Invention
The present invention relates to a method of manipulating eggs, particularly fertilized eggs.
b) Description of related art
The hard egg shell and large yolky egg of the avian embryo pose a significant obstacle to manipulating the embryo. When laid, the avian embryo consists of a blastoderm containing 30,000-60,000 cells on top of the yolk and encased in a hard calcified egg shell. Immediately below the shell is the egg shell membrane which surrounds the egg white, the egg yolk and the developing embryo. Many procedures, including transgenic modification of the avian genome, require access to the interior of the egg. For example, to modify the genetic material of a chicken, a small volume of liquid containing retroviral transducing particles or transfected donor cells must be injected into the subgerminal cavity of the recipient embryo. In addition, it may be desirable to expose the developing embryo to antigens, viruses, vaccines, or growth factors.
To provide access to the interior of the egg and the embryo, typically a hole or "window" is made in the egg shell. Petitte et al. (Development 108:185-89 (1990)) and Bosselman et al. (Science 243:533-35 (1989); U.S. Pat. No. 5,162,215) use a grinding tool, such as a Dremel, to grind a 5-8 mm hole in the egg shell. The underlying egg shell membrane is then cut away with a scalpel and 2-10 microliters of experimental solution is microinjected into the embryo. The hole is then sealed in one of several ways. Usually, the hole is covered with fresh egg shell membrane from a donor egg, with the membrane applied in the same orientation as in the egg, i.e, albumen-side down. When the membrane dries, it is permanently sealed with plastic model cement or a gas permeable surgical membrane. See also Carsience et al. (Development 117:669-75 (1993)), and Fraser et al. (Int. J. Dev. Biol. .37:381-85 (1993)).
Other similar methods have been used to access the developing embryo. Thoraval et al. (Transgenic Res. 4:369-76 (1995)) remove a triangular piece of shell, inject 10 ul of experimental solution through the opening into the embryo, then seal the egg by replacing the shell piece and covering it with adhesive tape. Marzullo (Nature 225:72-3 (1970)) cuts a hole in the shell, covers it with a glass cover slip, and seals it with paraffin wax.
The hatch rate of viable chicks following egg manipulations is an important concern, for often the objective of the egg manipulation process is the production of a genetically altered chick. For example, transgenic avians may be produced by injecting retroviral transducing particles or transfected donor cells into the embryo, and allowing the embryo to develop normally to hatching. As noted below, it is well known in the field that less than 10% of fertilized eggs hatch following manipulations that require opening of the shell. By contrast, greater than 90% of unmanipulated eggs will hatch if the eggs are from flocks that are at peak production. Work in several laboratories indicates that it is the opening procedure that decreases hatchability, not the injections.
Marzullo (1970) first reported the high mortality associated with opening the egg, noting that only 7% of embryos of windowed and injected eggs reached day 15 of incubation. Thoraval et al. (1995; Poultry Sci. 73:1897-1905 (1994)) also found that 2.3-7.3% of opened and injected eggs hatched; uninjected, windowed eggs had a similar hatching rate, suggesting that the opening procedure caused the low hatching rate. Petitte et al. (1990) reported that 4 out of 53, or 7.6%, of windowed and injected eggs hatched; hatchability was the same without injection, indicating that the windowing procedure per se was responsible for the low hatch rate. Although Bosselman et al. (1989) reported a hatch rate of 38% using essentially the same method as Petitte et al., consistently obtaining hatch rates over 10% continues to be problematic.
Surrogate shell methods have been developed to provide access to embryos and to improve hatch rates. Developing embryos, with or without genetic manipulation, are collected at various ages and transferred to "ex ovo" containers. Generally, 2 or 3 transfers are required as the embryo develops, and the last transfer consists of placing the embryo in a fresh donor egg shell with a large hole cut in the blunt end. Using these methods, Perry (Nature 331:70-73 (1988); U.S. Pat. No. 5,011,780) and Ono et al.(Dev. Biol. 161:126-30 (1994)) observed hatch rates greater than 25% for chicken and quail embryos, respectively. Unfortunately, this method is labor intensive and may prove rate-limiting if very large numbers of injections are necessary to produce viable transgenic chicks.
The surrogate shell method has also been combined with standard egg-opening methods. After windowing and injecting as described by Petitte et al. (1990), eggs are incubated for 3 days in standard incubators and the embryo is transferred to a surrogate shell which is sealed with gas permeable film as described in U.S. Pat. No. 5,011,780. This approach yields hatch rates of greater than 25%; however it is also labor intensive.
It would be desirable to provide an improved method for increasing the hatchability of eggs subjected to manipulation.